Robertsonian translocations in Paget's disease of bone.

The karyotypes of 14 patients with Paget's disease of bone were studied. The patients were recruited from our bone metabolism clinic where they received specific therapy for their skeletal disease. Eight of the 14 patients had chromosomal translocations localized to the D and G groups. None of the patients were related to one another, nor had any had the same lifelong environment. Thus, 57% of a sample of active patients with Paget's disease had Robertsonian translocations. By comparison, an age and sex-matched group of eight controls and 13 patients with osteoporosis who had been treated with bisphosphonates demonstrated no Robertsonian translocations. The prevalence of Robertsonian translocations in 14,000 newborns was reported to be 0.1%. These data suggest that a factor from the environment introduced during the lifetime of the patient could be present and could, in addition to genetic factors, affect gene replication during the development of Paget's disease.

Agar plate method using Lactobacillus plantarum for biotin determination in serum and urine.

An improved agar plate method of biotin bioassay using Lactobacillus plantarum ATCC 8014 and bromocresol purple was established to determine biotin levels in human serum and urine. Samples were treated with 4.5 N H2SO4 to liberate free biotin, autoclaved for 1 h and neutralized by 4.5 N NaOH, then 10 microliters was added to wells in each plate. The biotin levels were measured in 190 serum and 59 urine samples, and the means were 2.7 +/- 0.53 ng/ml and 12.4 +/- 5.56 ng/mg of creatinine, respectively. The intra-assay coefficient varience (CV) were 3.2 (n = 20) and 1.3% (n = 23), respectively. The recovery of biotin added (10 ng/ml) to serum was 110.7%, and to urine was 99.6%. These findings suggest that this assay is sufficiently accurate and reproducible for routine use in the clinical laboratory. The excretion of orally administered biotin was also demonstrated by the method.

The effects of biotin deficiency on organic acid metabolism: increase in propionyl coenzyme A-related organic acids in biotin-deficient rats.

Volatile organic acid levels in plasma and tissues and nonvolatile organic acid levels in urine of biotin-deficient (BD) rats were measured and compared with other factors of biotin deficiency. Biotin levels and the activities of propionyl coenzyme A (CoA) carboxylase (PCC) in the livers of these rats were decreased, respectively, to 22% +/- 3% and 3.6% +/- 0.3% of the average values of pair-fed controls. Plasma concentrations of propionate were higher (15 to 223 micrograms/mL) than those of controls (5 to 7 micrograms/mL), whereas plasma levels of 3-methylcrotonate were only minimally increased as compared with those of controls. Concentrations of these volatile acids in the tissues were similarly increased, although those in brain showed less remarkable increases as compared with levels in other tissues. In the urine of BD rats, large amounts of organic acids derived from propionyl CoA, as well as those from 3-methylcrotonyl CoA, were excreted. Plasma propionate levels were not apparently related to the severity of clinical symptoms, biotin levels, or carboxylase activities, but were related to the amounts of urinary ketone bodies, lactate, and some of the organic acids derived from branched-chain amino acids, including those from propionyl CoA.

Improved high-performance liquid chromatographic determination of biotinidase activity with biotinyl-6-aminoquinoline as substrate.

An improved high-performance liquid chromatographic assay method for biotinidase activity was developed using the fluorimetric substrate biotinyl-6-aminoquinoline, which was found to be more specific than the biotinyl-4-aminobenzoate previously used. The new method measures the intensity of the fluorescent signal at wavelengths (excitation 350 nm; emission 550 nm) longer than those (excitation 276 nm; emission 340 nm) for 4-aminobenzoate. The analysis of fluorescence in the visible spectrum reduced considerably the number of interfering peaks compared with analysis in the ultraviolet region. This method also made it possible to measure the biotinidase activity directly in samples usually difficult to calculate, such as human and bovine milk or porcine serum; the use of biotinyl-6-aminoquinoline allowed the analysis of the enzyme reaction in milk and porcine serum without pretreatment or dialysis. Stoichiometric increase and decrease of the substrate and product, respectively, were demonstrated. Michaelis constants for biotinyl-6-aminoquinoline were measured at various stages of partial purification. Because the solubility of these synthetic substrates in the aqueous phosphate buffer is limited, the determination of both Michaelis constant and maximum velocity by extrapolation may be helpful for the characterization of the kinetics of biotinidase.

Lipoamidase activity in normal and mutagenized pancreatic cholesterol esterase (bile salt-stimulated lipase).

Purified human milk lipoamidase was digested with endoproteinase Lys-C and the digested peptides were subjected to gasphase microsequence analysis. The sequencing of three isolated peptides of human milk lipoamidase revealed the identity of this protein with human milk bile salt-stimulated lipase (pancreatic cholesterol esterase). The identity of the cholesterol esterase with lipoamidase was confirmed by expressing a recombinant form of rat pancreatic cholesterol esterase and testing for lipoamidase activity of the recombinant protein. The results showed that the recombinant cholesterol esterase displayed both lipolytic and lipoamidase activities and was capable of hydrolysing triacetin and lipoyl-4-aminobenzoate (LPAB). The mechanisms of the esterase and amidase activities of the enzyme were further tested by determining enzyme activity in a mutagenized cholesterol esterase with a His435-->Gln435 substitution. This mutation has been shown previously to abolish enzyme activity against esterase substrates [DiPersio, Fontaine and Hui (1991) J. Biol. Chem. 266, 4033-4036]. We showed that the mutagenized protein was effective in hydrolysing the amidase substrate LPAB and displayed similar enzyme kinetics to those of the native enzyme. These data indicate that the mechanism for the cholesterol esterase hydrolysis of lipoamides is different from that of the hydrolysis of substrates with an ester linkage. The presence of an enzyme in the gastrointestinal tract capable of both ester and amide hydrolysis suggests an important role for this protein in the digestion and absorption processes.

Biocytin-specific 110-kDa biotinidase from human serum.

Biotinidase purification from human serum was performed under new protocol. With HPLC biotinidase assay instead of colorimetric method and using non-ionic surfactant, 110-kDa biotinidase was discovered and co-purified in addition to the previously identified 76-kDa biotinidase. This newly identified enzyme accounted for 5% of the total biotinidase activity. Protein core of 110 kDa was estimated as 72 kDa by use of N-glycanase and SDS-PAGE analysis, while that of the 76-kDa enzyme was estimated as 59 kDa. Total amino acid analysis indicated 30% higher absolute amounts of amino acids in 110-kDa enzyme. The following differences were observed from kinetic study: the 110-kDa enzyme showed a 10-fold lower Km value and a 9-fold higher kcat/Km value for biocytin than those of 76-kDa enzyme. Thus, 110-kDa enzyme is more likely to be the physiological biocytin hydrolase (biocytinase), since the biocytin concentration in human serum is extremely low. The pathogenesis of an inborn error of the metabolism such as a variant form of biotinidase deficiency, which presented an atypical clinical course, might be related to these isoenzymes in terms of their different roles in the body.

Biocytin synthetase activity in human milk as assessed by high-performance liquid chromatography.

A reversed-phase liquid chromatographic assay for biocytin synthetase activity has been developed. By this method, biocytin synthetase, isolated to homogeneity from human milk, was found to synthetize biocytin from biotin and L-lysine in the presence of ATP and magnesium ion(s). Both ATP and magnesium ion(s) were required for the synthesis of biocytin. Equal molar amounts of ADP and ATP were produced and consumed, respectively, in the course of the production of the same molar amount of biocytin; however, production of AMP was not observed. Biocytin synthetase Michaelis constants were 2.5, 1.8, and 0.11 mM for biotin, L-lysine, and ATP, respectively. Biocytin synthetase from milk was shown to synthesize biocytin in a stoichiometric amount.

Release of anchored membrane enzymes by lipoamidase.

Lipoamidase is found to be able to release various membrane-anchored enzymes from the membrane compartment of pig brain. Released enzymes revealed their intact enzyme activities in the soluble fraction. Lipoamidase could release at least two types of anchored enzymes, i.e. glycosyl-phosphatidylinositol-bonded and myristoylated enzymes, but not integral membrane bound enzymes. The reaction was competitively inhibited by lipoyllysine. This releasing mechanism found in the membranes may play important roles in the secretory mechanism of extracellular enzymes and also in the cellular signal-transduction system through topological changes in cellular enzymes.

Enkephalin hydrolysis by human serum biotinidase.

Purified human serum biotinidase exhibited amino-exo-peptidase activity. Enkephalins and dynorphin A (less than 10-mer) seemed to be the most appropriate substrates among various physiological peptides in terms of the kcat/Km values. Similar kcat/Km values were obtained for both biocytin (biotinyllysine) and these opioid-neuropeptides. Neuro-oligo-peptides ranging from 2-mer to 18-mer were hydrolyzed. The presence of amino group at the carboxyl terminal position increased the kcat/Km value by decreasing the Km value. The results of inhibition studies using various kinds of antibiotic inhibitors, metals, and chelating agents indicated that enkephalin hydrolysis was mediated by the peptide-hydrolyzing center probably containing Zn ions. This aminopeptidase activity was uniquely inhibited by a vitamin of biocytin. The reason for the high content of biotinidase activity in serum may be related to the binary function of this enzyme; i.e., biocytin hydrolyzing amidase and enkephalin hydrolyzing aminopeptidase functions.

Lipoamidase is a multiple hydrolase.

The substrate specificity of lipoamidase, purified from the pig brain membrane with lipoyl 4-aminobenzoate (LPAB) as a substrate, was extensively studied. This single polypeptide was found to hydrolyse the bonding between amide, ester and peptide compounds. However, stringent structural requirements were found in the substrates, e.g. LPAB was hydrolysed, whereas biotinyl 4-aminobenzoate was not, as stated in our previous paper [Oizmui & Hayakawa (1990) Biochem. J. 266, 427-434]. The enzyme specifically recognized the whole molecular structure of the substrate, whereas it loosely recognized the bond structure of the substrate; e.g. the dipeptide Asp-Phe was not hydrolysed, whereas the methyl ester of Asp-Phe (aspartame) was. The exopeptidase activity was demonstrated by lipoamidase; however, longer peptides than the hexamer seemed not to be substrates. Lipoyl esters, which were electrically neutral, exhibited higher specificity with longer acyl groups. Molecular mass and molecular hydrophobicity (hydropathy) seemed to determine the substrate specificity. Lipoyl-lysine, acetylcholine and oligopeptides were hydrolysed at similar Km values; however, acetylcholine was hydrolysed at a velocity 100 times higher. Although many similar specificities were found between electric eel acetylcholinesterase and lipoamidase, distinctly different specificity was demonstrated with lipoyl compounds. The role of lipoamidase, which resides on the brain membrane and possesses higher specificity for hydrophobic molecules, remains to be elucidated.

High-performance liquid chromatographic determination of four kinds of biotin-containing carboxylase.

A convenient and reliable reversed-phase liquid chromatographic assay for four kinds of carboxylase, i.e. pyruvate carboxylase, acetyl-coenzyme A carboxylase, propionyl-coenzyme A carboxylase, and 3-methylcrotonyl-coenzyme A carboxylase, was developed. These are all biotin enzymes, which contain covalently bound biotin as a co-factor (co-enzyme). This chromatographic method, which is free from interferences, gives a stoichiometric and precise assay for these enzymes. It is non-radioisotopic, which makes it suitable for clinical laboratories. The assay time required for one sample is ca. 30 min. The method was satisfactorily applied to guinea-pig kidney homogenates after a simple pretreatment with acid.

Biotinidase in the porcine cerebrum.

Biotinidase activities found in porcine brains (n = 3) were as follows: cerebrum, 4.4 +/- 0.2 pmol/min per milligram of protein; cerebellum, 7.6 +/- 0.3 pmol/min per milligram of protein; medulla, 2.9 +/- 0.3 pmol/min per milligram of protein. These values are relatively high compared with the activities in rat or guinea pig brains. Subcellular distribution of biotinidase was found mainly in the soluble cytoplasmic fraction (S3), i.e., in the supernatant of 0.32 M sucrose S2 solution after ultracentrifugation at 105,000g for 90 min. This is in contrast to the guinea pig livers, in which the subcellular distribution of biotinidase is mainly found in the microsomal fraction. After a seven-step purification (22,200-fold enrichment), porcine brain biotinidase is identified as a single polypeptide by the sodium dodecyl sulfate-polyacrylamide gel electrophoresis system, and its molecular weight is determined as 68,000 Da. The isoelectric point of the enzyme was 4.3. Sialidase treatment strongly suggests the presence of sialyl residues in this enzyme. Amino acid analysis indicates relatively high hydrophilicity and high content of glycine and serine. The enzyme activity is inhibited by organic mercurials, but not by diisopropylfluorophosphate. Abundant soluble biotinidase in brain cytoplasm may play an important role which has not been discovered yet.

[Clinical evaluation of serum biotin levels and biotinidase activities in patients with various liver diseases].

In order to evaluate the clinical significance of serum biotin and biotinidase in liver disease, serum biotin levels and biotinidase activities were determined in 83 patients with various liver diseases and 10 healthy controls. Serum biotin levels and biotinidase activities were determined by a simplified lactobacillus plantarum bioassay and liquid chromatography with fluorimetric detection respectively. Serum biotin levels in decompensated liver cirrhosis, hepatoma and fulminant hepatitis were found to be significant low compared with healthy controls, while it was significant high in autoimmune hepatitis. There was no significant difference between serum biotin levels in the other liver diseases and healthy controls. In various liver diseases except for both acute hepatitis and alcoholic liver disease biotinidase activities were significantly reduced than in healthy controls. Serum biotinidase activities were correlated with serum albumin, prothrombin time, ChE and total cholesterol respectively, suggesting that biotinidase activities may reflect the degree of liver damage. These results seem that biotin deficiency may occur in some cases of severe liver diseases.

Effect of phospholipids on purified lipoamidase.

Phospholipids were added to purified lipoamidase from porcine brain microsomal membranes, and changes in lipoamidase activity were examined. Approximately twofold activation of lipoamidase activity occurred upon the addition of phosphatidylethanolamine. On the other hand, phosphatidylserine, cardiolipin, and phosphatidic acid reduced the enzyme activity by approximately 80%. This pattern of the activation of lipoamidase by phosphatidylethanolamine and its inhibition by phosphatidylserine is similar to the pattern for adenylate cyclase, and contrasts with the pattern for ATPase.

Rothmund-Thomson syndrome associated with trisomy 8 mosaicism.

This report describes a boy with Rothmund-Thomson syndrome associated with trisomy 8 mosaicism. The patient presented with typical features of Rothmund-Thomson syndrome but some of the features often seen in trisomy 8 mosaics were also observed in him. The possibility that the two disorders might share a common pathogenesis is postulated.

Lipoamidase (lipoyl-X hydrolase) from pig brain.

Although the optimum substrate for lipoamidase (lipoyl-X hydrolase) has not yet been determined, it is known that lipoamidase activity, as determined by hydrolysis of the synthetic substrate lipoyl 4-aminobenzoate (LPAB), is widely distributed in pig brain tissues, i.e. in the cerebrum, cerebellum and medulla. Over 95% of the enzyme activity is present in the membrane subfractions, indicating that brain lipoamidase is an integral membrane protein enzyme. To elucidate the chemical nature and the optimum substrate of the abundant lipoamidase in the brain, we isolated it from the membrane subfractions. After an 8-step purification procedure, brain lipoamidase was purified 601-fold and identified as a 140 kDa glycoprotein by SDS/PAGE. A mechanistic study to determine Km and Vmax, values was carried out using various synthetic compounds. Lipoyl-lysine, which is generally believed to be a naturally occurring substrate of lipoamidase, was first compared with biotinyl-lysine, because these two vitamins have reactive sulphur atoms and are similar in molecular mass and structure. The Km for lipoyl-lysine was 333 microM, whereas biotinyl-lysine was not hydrolysed. Stringent specificity for the lipoyl moiety is demonstrated, as expected. Dipeptides of amino acid-lysine structures were studied, and dipeptides of aspartyl- and glutamyl-lysine hydrolysis occurred at high Km (3 mM) values. Thus lysine in the moiety is not very effective as an optimum substrate. The chemical bond structures of the amide bond (lipoyl-lysine) and peptide bond (aspartyl-lysine) were hydrolysed. Next, the ester bond compound was tested, and it was observed that lipolylmethyl ester was hydrolysed at high specificity. These findings indicate that this enzyme has broad specificities with respect to bond structure; it therefore is a unique hydrolase having stringent specificity for lipoic acid and relatively broad specificity for the chemical bond and the X moiety. Various inhibitors were tested; a few reagents, such as organic mercurials, di-isopropylfluorophosphate, 1,10-phenanthroline, sodium azide and angiotensin-converting enzyme inhibitor exhibited some inhibition (not more than 60%). Thus the active centre of this enzyme is a complex type. Although ATP is not hydrolysed and the lowest Km value is exhibited by the synthetic substrate reduced from LPAB (12 microM), some other compounds may still be expected to be hydrolysed by this unique and abundant brain lipoamidase.

Interaction of metals at the reaction center of lipoamidase.

Various metals have been shown to inhibit porcine brain lipoamidase activity at 0.1 mM, but not ferrous and ferric ions. However, in the presence of ethylenediamine tetraacetic acid (EDTA) 0.1 mM iron ions did inhibit the activity. No other metals exhibited this type of increased inhibition with the addition of EDTA. The ferric- and ferrous-EDTA compounds were equally effective. Various Fe-containing compounds also inhibited the enzyme activity, the order of inhibition being: EDTA greater than o-phenanthroline greater than azide greater than citrate. Hemin also inhibited the enzyme activity strongly. However, Fe-proteins, e.g. cytochrome c, transferrin and peroxidases, were not inhibitory. These results indicate the importance of Fe ion chelates with structural and molecular size differences for interaction with the reaction center of this enzyme.